It is now known that single and double-stranded RNA can modulate expression of or modify processing of target RNA molecules by a number of mechanisms. Some such mechanisms tolerate variation in the amount of sequence complementarity required between the modulatory (or interfering) RNA and the target RNA. Certain microRNAs can translationally repress target mRNA having as little as 6 nucleotides of complementarity with the microRNA. The development of RNA interference agents, for example, using double-stranded RNA to repress expression of disease-related genes is currently an area of intense research activity.
Double-stranded RNA of 19-23 bases in length is recognized by an RNA interference silencing complex (RISC) into which an effector strand (or “guide strand”) of the RNA is loaded. This guide strand acts as a template for the recognition and destruction of highly complementary sequences present in the transcriptome. Alternatively, through the recognition and binding of RNA sequences of lower complementarity, interfering RNAs may induce translational repression without mRNA degradation. Such translational repression appears to be a mechanism of action of endogenous microRNAs, a group of short non-coding RNAs involved in differentiation and development.
Efforts at developing interfering RNAs for therapeutically applications thus far have focused on producing specific double-stranded RNAs, each with complete complementarity to a particular target transcript. Such double-stranded RNAs (dsRNAs) are potentially effective where a single suitable target can be identified. However, dsRNAs, particularly those designed against one target, may have at least two categories of off-target side effects that need to be avoided or minimized. Undesirable side effects can arise through the triggering of innate immune response pathways (e.g. Toll-like Receptors 3, 7, and 8, and the so-called interferon response) and through inadvertent inhibition of protein expression from related or unrelated transcripts (either by RNA degradation, translational repression or other mechanisms). Inadvertent side-effects can be obtained when the passenger strand of a duplex is loaded and generates suppression of RNA species distinct from those targeted by the putative guide strand. Loading bias is well understood and most design processes only select sequences for a RNAi duplex from which only the intended guide strand will be loaded. Thus, some bioinformatic and/or experimental approaches have been developed to try to minimize off-target effects. Algorithms for in silico hybridization are known, and others have been developed for predicting target accessibility and loading bias in an effort to eliminate or minimize side-effects while maintaining effectiveness.
Several double-stranded RNA molecules for potentially treating human diseases of viral and non-viral origin are in various stages of development. The diseases include Age-related Macular Degeneration, Amyotrophic Lateral Sclerosis (ALS), and Respiratory Syncytial Virus (RSV) infection. These RNA molecules, however, only target a single site in an RNA sequence. Although RNA interference may be useful and potent in obtaining knock-down of specific gene products, many diseases involve complex interactions between ontologically-unrelated gene products. Thus, the use of single-gene targeting approaches may not succeed except where a single or dominant pathophysiologic pathway can be identified and interrupted.
In fact, many putative targets can be identified for most diseases. Attempts to confirm that inhibiting single targets in isolation is therapeutically valuable have been disappointing.
Indeed, obtaining therapeutic effectiveness is proving to be extremely challenging, probably because of multiple levels of redundancy in most signaling pathways. For example, many disorders, such as cancer, type 2 diabetes, and atherosclerosis, feature multiple biochemical abnormalities. In addition, some putative targets may be subject to enhanced mutation rates, thereby negating the effects of interfering RNAs on any such target.
For example, therapeutic approaches to viral infections continue to be major challenges in agriculture, as well as in animal and human health. The nature of the replication of viruses makes them highly plastic, “moving targets” therapeutically—capable of altering structure, infectivity, and host profile. The recent emergence of viruses such as Severe Acute Respiratory Syndrome (“SARS”) and Avian Influenza Virus (“bird flu”) exemplify these challenges. Even well-described viruses such as those involved in Acquired Immunodeficiency Syndrome or AIDS (e.g. Human Immunodeficiency Viruses, HIV-1 and HIV-2), continue to defy efforts at treatment and vaccination because of on-going viral mutation and evolution.
Furthermore, although nucleic acid therapeutics such as interfering RNAs are candidates for viral therapy, in part because modern rapid gene sequencing techniques allow viral genome sequences to be determined even before any encoded functions can be assessed, the error-prone replication of viruses, particularly RNA viruses, means that substantial genomic diversity can arise rapidly in an infected population. Thus far, strategies for the development of nucleic acid therapeutics have largely centered on the targeting of highly-conserved regions of the viral genome. It is unclear whether these constructs are efficient at treating viral infection or preventing emergence of resistant viral clones.
Therapeutic approaches that involve the design and use of one interfering RNA for control of several key “drivers” of the disease are thus desirable. Therefore, there is a need for interfering RNAs which can modulate multiple RNAs or target multiple sites within an RNA. Methods for the design and for making such therapeutic multi-targeting interfering RNAs are also needed. Antiviral interfering RNAs that can be developed rapidly upon the isolation and identification of new viral pathogens and that can be used to help slow, or even prevent, the emergence of new, resistant isotypes are also needed. Finally, it would be useful to have such RNAs wherein each of the two strands of a synthetic duplex independently targets at least one of the multiple target RNAs.